Investigating calcification-related candidates in a non-symbiotic scleractinian coral, Tubastraea spp.

In hermatypic scleractinian corals, photosynthetic fixation of CO2 and the production of CaCO3 are intimately linked due to their symbiotic relationship with dinoflagellates of the Symbiodiniaceae family. This makes it difficult to study ion transport mechanisms involved in the different pathways. In contrast, most ahermatypic scleractinian corals do not share this symbiotic relationship and thus offer an advantage when studying the ion transport mechanisms involved in the calcification process. Despite this advantage, non-symbiotic scleractinian corals have been systematically neglected in calcification studies, resulting in a lack of data especially at the molecular level. Here, we combined a tissue micro-dissection technique and RNA-sequencing to identify calcification-related ion transporters, and other candidates, in the ahermatypic non-symbiotic scleractinian coral Tubastraea spp. Our results show that Tubastraea spp. possesses several calcification-related candidates previously identified in symbiotic scleractinian corals (such as SLC4-γ, AMT-1like, CARP, etc.). Furthermore, we identify and describe a role in scleractinian calcification for several ion transporter candidates (such as SLC13, -16, -23, etc.) identified for the first time in this study. Taken together, our results provide not only insights about the molecular mechanisms underlying non-symbiotic scleractinian calcification, but also valuable tools for the development of biotechnological solutions to better control the extreme invasiveness of corals belonging to this particular genus.

In hermatypic scleractinian corals, photosynthetic fixation of CO 2 and the production of CaCO 3 are intimately linked due to their symbiotic relationship with dinoflagellates of the Symbiodiniaceae family. This makes it difficult to study ion transport mechanisms involved in the different pathways. In contrast, most ahermatypic scleractinian corals do not share this symbiotic relationship and thus offer an advantage when studying the ion transport mechanisms involved in the calcification process. Despite this advantage, non-symbiotic scleractinian corals have been systematically neglected in calcification studies, resulting in a lack of data especially at the molecular level. Here, we combined a tissue micro-dissection technique and RNA-sequencing to identify calcification-related ion transporters, and other candidates, in the ahermatypic non-symbiotic scleractinian coral Tubastraea spp. Our results show that Tubastraea spp. possesses several calcification-related candidates previously identified in symbiotic scleractinian corals (such as SLC4-γ, AMT-1like, CARP, etc.). Furthermore, we identify and describe a role in scleractinian calcification for several ion transporter candidates (such as SLC13, -16, -23, etc.) identified for the first time in this study. Taken together, our results provide not only insights about the molecular mechanisms underlying non-symbiotic scleractinian calcification, but also valuable tools for the development of biotechnological solutions to better control the extreme invasiveness of corals belonging to this particular genus.
In scleractinian corals (Cnidaria, Anthozoa), also known as stony corals, calcification leads to the formation of a biomineral composed of two fractions, one made of calcium carbonate (CaCO 3 ) in the mineral form of aragonite [1][2][3] , and the other made of organic molecules [4][5][6] . Based on the ability of scleractinian corals to build reef structures, they are functionally divided into two main groups, namely, hermatypic (i.e. reef-building) and ahermatypic (i.e. non-reef-building). The majority of hermatypic corals hosts symbiotic dinoflagellates of the Symbiodinacae family 7 in their tissues, commonly known as zooxanthellae 8 . This symbiotic association, which is lacking in most ahermatypic corals, provides the nutritional foundation for the host metabolism and boosts calcification in nutrient-poor tropical waters 9 .
Given the ability of hermatypic corals to build reefs, and given the economic and ecological importance associated with reef structures 10 , symbiotic scleractinian corals have been a major focus of calcification research over the years 2,11 . Whereas, ahermatypic non-symbiotic scleractinian corals have not been extensively studied and to date they remain under-represented especially in terms of molecular data 12 . These corals, however, should not be neglected as they represent important resources for scleractinian calcification research. This is because, in symbiotic scleractinian corals, calcification is linked to the photosynthetic fixation of CO 2 -both at the spatial as well as the temporal scales-which makes it difficult to disentangle these processes. Whereas, non-symbiotic scleractinian corals allow studying the transport mechanisms involved in calcification without the confounding factor of symbiosis 13 . In addition, studying calcification in non-symbiotic scleractinian corals further allows obtaining comparative information on the different scleractinian calcification strategies, therefore aiding in a better understanding of how calcification evolved within this order.
One of the main questions surrounding scleractinian calcification is how (i.e. via which molecular tools) corals promote a favorable environment for calcification 14 . As in other biological groups, coral calcification is a biologically controlled process, meaning that the precipitated mineral is not a byproduct of metabolic processes (also known as biologically induced biomineralization), but rather under strict biological and physiological control 15,16 . This control is exerted by a specialized tissue called the calicoblastic epithelium, that comprises the calcifying calicoblastic cells 17 . These cells control and promote calcification by modifying the chemical composition at the sites of calcification, which comprise intracellular vesicles and the extracellular calcifying medium (ECM) 14 . As recently suggested, calcification begins with the formation of amorphous calcium carbonate (ACC) nanoparticles, within intracellular vesicles, in the calicoblastic cells. ACC nanoparticles are then released via exocytosis into the ECM 18 . Here, ACC nanoparticles attach (i.e. nanoparticle attachment) and crystallize, while ions fill the interstitial spaces between them (i.e. ion-by-ion filling). Both, nanoparticle attachment and ion-byion filling processes require the calicoblastic cells to regulate ion transport and their concentration at the sites of calcification 2,19 . Furthermore, the calicoblastic cells also secrete an organic matrix which may stabilize ACC in the intracellular vesicles and play other roles, such as aiding and promoting ACC crystallization [20][21][22][23][24] .
Ion (i.e. calcium, carbonate, protons, and others) transport, to and from the sites of calcification, is of particular interest 2,19 . For instance, calcium and carbonate ions, the building blocks of the coral skeleton, have to be constantly supplied to the sites of calcification to sustain its growth 25 . Whereas, protons must be removed from the sites of calcification to increase the aragonite saturation state, prevent dissolution of calcium carbonate nanoparticles, and promote ion-by-ion filling mechanisms 14 .
Over the years, the ion transport model underlying scleractinian calcification has been well characterized for the calicoblastic cells through physiological and molecular studies [26][27][28] . However, such understanding is only partial and many calcification-related ion transporters still need to be identified. When searching for calcificationrelated candidates, different approaches are possible. One is the so called "targeted" approach and is based on the analysis of genes and/or proteins that have been chosen a priori-generally based on known biological functions in other model systems. This approach is extremely powerful for studying the genetic architecture of complex traits, such as calcification, in addition to being an effective approach for direct gene discovery 29 . Nevertheless, although the targeted approach has led to the identification of some of the most relevant calcification-related candidates in scleractinian calcification 13,[30][31][32] , it is largely limited by the requirement of existing knowledge about the gene(s) under investigation. To overcome this limitation, other approaches, the so-called "broad" approaches, have been developed throughout the years. Broad approaches have the potential to discover novel candidates and pathways that have not been previously considered in the context of calcification, thus allowing a more holistic understanding of the process. These approaches have been performed at different levels, including the transcriptomic one, which relies on the use of RNA-sequencing (RNA-seq) technology [33][34][35] . To date, however, the use of RNA-seq to identify calcification-related candidates has been limited to analyzing coral molecular responses to environmental parameters known to influence calcification (such as light 33 and CO 2 36 ), and only one study, performed in the symbiotic scleractinian coral Stylophora pistillata, has analyzed genes being more highly expressed in the coral calcifying tissue 37 .
Therefore, given the high potential of broad approaches in discovering novel candidates, and given the scarce amount of data available for non-symbiotic scleractinian corals 12 , we have performed, in this study, RNA-seq on coral species belonging to the ahermatypic non-symbiotic scleractinian genus Tubastraea (Lesson, 1829) 38 . Tubastraea corals include invasive saltwater species 39-42 that were introduced into the southwestern Atlantic on oil platforms 42 . Since the late 1980s, these corals have been colonizing the rocky shores of the southeastern Brazilian coast 40 . Their rapid spread and growth provides them a competitive advantage and, therefore, represent a serious risk for endemic biodiversity loss 43 . In the absence of innovation in control methods, the dispersal of Tubastraea is expected to continue. In this context, calcification studies are fundamental to a better understanding of the life histories and population ecology of this genus. Of particular interest is the rapid linear skeletal growth of Tubastraea that could increase the competitiveness of these species 44 . In this study, we searched for calcification-related candidates, by sequencing the whole transcriptome from total colonies and oral fractions (i.e. fractions devoid of the aboral tissues that contain the calicoblastic cells) of Tubastraea spp., obtained through a tissue micro-dissection technique. After assembling and annotating a highly complete transcriptome for Tubastraea spp., we identified and analyzed genes enriched in the total colony transcriptomes compared to the oral fraction transcriptomes. The analysis included both a comparison with calcification-related candidates previously characterized in symbiotic scleractinian corals, as well as a search for novel calcification-related ion transporter candidates.
This study provides insights into the molecular mechanisms underlying non-symbiotic scleractinian calcification and identifies valuable tools for the development of biotechnological solutions to better control the extreme invasiveness of corals belonging to this genus.

Results
Sequence read data and raw data pre-processing. RNA sequencing was performed for two sample groups, total colony and oral fraction (i.e. fraction devoid of the aboral tissues containing the calcifying calicoblastic cells), of three independent biological replicates (n = 3) of Tubastraea spp. Both groups, obtained through a previously developed micro-dissection protocol 45 , produced a total of 539,331,300 raw reads with an average of 44.9 ± 8.7 (mean ± SD) million read pairs per sample. Raw reads were subjected to quality trimming, which included adaptor removal, yielding a total of 369,357,576 trimmed reads.
De novo transcriptome assembly and quality assessment. Trimmed reads were subjected to de novo whole transcriptome assembly using Trinity, after being further reduced to 73

Functional annotation.
To evaluate the completeness of the transcriptome library, functional annotation-including GO terms, EggNOG and KEGG pathway enrichment analysis-of the whole transcriptome of Tubastraea spp. was performed using Blastx results and OmicsBox. A summary of the whole transcriptome assembly and annotation results is listed in Table 1.

Differential expression analysis.
To identify differentially expressed genes between the total colony and the oral fraction, we first selected genes that had count per millions (CPM) more than 1 in at least two samples. Differential expression analysis was then performed using OmicsBox, followed by Benjamini-Hochberg multiple test correction. A total of 4,483 genes were reported to be differentially expressed (FDR < 0.05, LogFC < ± 1) between the total colony and the oral fraction (Table 1). Of these, 3,174 genes were significantly enriched in the total colony compared to the oral fraction, and 1,309 genes were significantly enriched in the oral fraction compared to the total colony. Differentially Expressed Genes (DEGs) have been clustered using Pearson's correlation and displayed in a heatmap (Fig. 2). In this heatmap, biological replicates (1, 2 and 3) show strong clustering  www.nature.com/scientificreports/ within the same group (Total and Oral), which are also clearly separated. In addition, a Multi-Dimensional Scaling (MDS) plot was performed to examine the homogeneity across biological replicates (Fig. 3). According to the MDS results, biological replicates showed strong clustering within each group and each group formed a distinct cluster.
Our results also show that 34 (62%) of the 55 protein sequences are not differentially expressed between the total colony and the oral fraction, and thus are not found in the heatmap (Fig. 4). These included: H v CNs, SLC9s, PMCAs, and VGCC. www.nature.com/scientificreports/ Functional annotation and identification of unigenes putatively involved in coral calcification. DEGs were annotated using the same databases used for the whole transcriptome annotation. First, GOterm enrichment analysis using Fisher's exact test was performed to infer which biological processes are associated with the enriched genes in the total colony compared to the oral fraction. Our results show 13 enriched GO-terms, including biological processes associated with "carbohydrate metabolic process", "extracellular space", "cell adhesion", "extracellular matrix" and "extracellular matrix organization" (Fig. 5). EggNOG functional annotation of the enriched genes in the total colony compared to the oral fraction was then performed. A total of 2,841 out of 3,174 enriched genes (88.6%) are functionally annotated into 23 COG functional categories, including inorganic ion transport and metabolism (P), and intracellular trafficking, secretion and vesicular transport (U) (Fig. 6).  www.nature.com/scientificreports/ Finally, using the KO term, provided by the EggNOG mapper, of each annotated gene, we performed KEGG annotation. KEGG annotation further divided genes into multiple families. Among these, 39 KO terms are associated with ion transporters (Table 2).

Discussion
The "calcification toolkit" is the collective term documented and/or hypothesized to be involved in biomineral formation at various stages of an organism's life history 52 . Out of all the toolkit components, proteins have been the most intensively characterized 26,53,54 . As a result, proteomic studies have suggested that, although proteins from distant organisms share common properties 53 , each taxon-specific suite appears to have evolved independently through convergent and co-option evolution. This has led to variable contributions, from new lineage-and species-specific proteins, to the "calcification toolkit", which show contrasting rates of conservation between and   www.nature.com/scientificreports/ within lineages 55 . Several tools of the "calcification toolkit" have also been identified in scleractinian corals 48,56 , yet to date only few experiments have been conducted and solely for symbiotic species 54 . Other than being particularly attractive for calcification studies because of the lack of symbiotic dinoflagellates in their tissues, corals belonging to the Tubastraea genus have been the focus of numerous biological [57][58][59] and ecological research studies 60,61 aiming at identifying key parameters underlying their invasiveness. Nevertheless, their "calcification toolkit", which may include specific components providing these corals with an advantage in terms of calcification strategies, has never been investigated at the molecular level. In this study, we aimed to fill this knowledge gap by searching for candidates of the "calcification toolkit" in the non-symbiotic scleractinian coral Tubastraea spp. using a tissue micro-dissection technique to remove the oral fraction (easily accessible and free of the calicoblastic cells) from the total colony of Tubastraea spp. This previously developed technique has already been used in the past and has contributed to the identification of some of the most frequently searched and studied candidates in a wide range of calcifying metazoans 26,62,63 , other than corals 31,45,47 . By coupling this technique with RNA-seq, we have then identified and analyzed differentially expressed genes with a focus on those enriched in the total colony compared to the oral fraction. Indeed, these genes are specific of the aboral tissues and include calicoblastic cell-specific genes, that could play a role in calcification. This is supported by our results showing that, although many genes are ubiquitously expressed in the total colony-and thus in both oral and aboral tissues -, others are differentially expressed, with clearly distinct expression profiles between the total colony and the oral fraction ( Figs. 2 and 3). It follows that the different expression profiles reflect specific gene functions related to the oral and aboral tissues. Amongst the 3,174 aboral-specific genes (Table 1), we identified most calcification-related candidates previously described as part of the "calcification toolkit" of symbiotic scleractinian corals (Fig. 4). These include the bicarbonate transporter SLC4-γ 64 . SLC4-γ has been proposed to play a role both in the regulation of intracellular HCO 3 homeostasis-which is critical to buffer excess of H + generated during CaCO 3 precipitation-and the supply of HCO 3 to the calcifying cells in several organisms, including sea urchins 63,65 , mussel 62 , coccolithophores 66 and corals 31,67 . We also identified an ammonium transporter belonging to the AMT1 sub-clade (Fig. 4). AMT transporters have been suggested to play a role in calcification in multiple metazoans, including mollusks [68][69][70] and symbiotic scleractinian corals [71][72][73] . Although their role in coral calcification still needs to be investigated in detail, it has been suggested that AMT1 transporters mediate pH regulation in the ECM by transporting NH 3 into the ECM which could buffer excess of protons. Organic matrix proteins, including 2 CARPs and 1 SCRiP, were also identified (Fig. 4). CARPs are proteins with dominant Low Complexity Domains (LCDs) that have been described in the secreted organic matrix of biominerals in different metazoan taxa [74][75][76][77] . CARPs have been identified also in previous proteomic studies on coral skeletons 35 , where they have been suggested to play a role in CaCO 3 formation given their high affinity to positively charged ions (i.e. Ca 2+ ) [78][79][80] . SCRiPs, instead, are a family of putatively coral-specific genes for which different roles have been suggested based on their molecular features (i.e. presence of signal peptide, high amino acidic residues content and cysteine-rich) 50 . Moreover, three galaxins-like proteins and three CAs were also identified (Fig. 4). Galaxin was first identified in the exoskeleton of the scleractinian coral Galaxea fascicularis and was described as a tandem repeat structure with a di-cysteine motif fixed at nine positions 49 . Since this discovery, galaxin homologs have been observed in the exoskeleton of other scleractinian species [81][82][83] , as well as in mollusks 84 and squid 85 . It has also been shown that galaxin is associated with the developmental onset of calcification after larval stage in Acropora millepora 86 . Whereas CAs are metallo-enzymes that catalyze the reversible hydration of CO 2 into HCO 3 -, the source of inorganic carbon for CaCO 3 precipitation. In metazoans, CAs belong to a multigenic family and are widely known to be involved in calcification in diverse metazoans such as sponge spicules 87 www.nature.com/scientificreports/ been suggested to play a direct role in calcification 13 . Here, we have identified 3 CAs with higher expression in the total colony compared to the oral fraction, which suggests a potential role in calcification. The presence of these candidates amongst the aboral-specific genes of a non-symbiotic scleractinian coral strongly suggests a calcification-related function and, in a wider context, further supports the hypothesis of a "common calcification toolbox" in scleractinian corals, as previously suggested 93 .
However, several components of the toolbox have not been identified amongst the aboral-specific genes of Tubastraea spp. (Fig. 4). These include: (1) voltage-gated H + channels (H v CN), that have been suggested to participate in the pH i homeostasis of calcifying coccolithophore cells 94 , in the larval development and shell formation of the blue mussel 62 and in the calicoblastic cells of several symbiotic scleractinian coral species 47,95 ; (2) SLC9s, that have been suggested to play a role in H + removal during trochophore development in mussels 62 and coral calcification 47 , (3) Plasma Membrane Ca 2+ -ATPase (PMCA), which have been suggested to take part in Ca 2+ supply to the sites of calcification in mussels 62 , as well as in the pH ECM regulation of the ECM in corals 30 , neurexins, that connect the calicoblastic cells to the extracellular matrix in corals 48 and (4) Voltage-Gated Ca 2+ -channels (VGCC), which have been suggested to facilitate Ca 2+ transport in the calcifying epithelium of oysters 96 and corals 30 . One possible explanation of these results is that gene gain/loss or even change of protein function has occurred during scleractinian evolutionary history, resulting in a different "calcification toolkit". This is further supported by the hypothesis that, although calcification-related proteins from distant organisms share common properties 53 , they have evolved independently, through convergent evolution and co-option, in each taxon, thus resulting in contrasting rates of conservation between and within lineages 55 .
In addition to comparing these calcification-related candidates between non-symbiotic and symbiotic scleractinian corals, we have also searched for novel ion transporter candidates of the "calcification toolkit" in Tubastraea spp. by focusing on the rest of the aboral-specific genes. To explore their involvement in biological processes, we performed a GO enrichment analysis, and showed that, among several processes, these aboralspecific genes are enriched in "extracellular space", "cell adhesion", "extracellular matrix" and "extracellular matrix organization" (Fig. 5). These results highlight the importance of extracellular matrices in the aboral tissues, in which they play a pivotal role in the spatial organization of the cells, as they organize them according to their function. Some examples include both the organic extracellular matrix (ECM), which facilitates cell-cell and cellsubstrate adhesion with the help of desmocytes 97 , as well as the skeletal organic matrix (SOM), which facilitates the controlled deposition of the CaCO 3 skeleton. Also, the expression of genes in the aboral tissues linked to "carbohydrate metabolic processes" suggests an enrichment of biochemical processes involved in carbohydrate metabolism, which may ensure a constant supply of energy needed to support the energy-demanding process of calcification 98,99 .
Moreover, EggNOG annotation (Fig. 6) shows that some of the aboral-specific genes belong to the following categories: "inorganic ion transport and metabolism" and "intracellular trafficking, secretion, and vesicular transport", thus underling the importance of membrane and vesicular transport linked to calcification in the aboral tissues 2,14,18 . These results are supported by recent observations of intracellular vesicles moving towards the calcification site both in corals 14,18 and sea urchins 100 . Calcification-related ions have been suggested to be highly concentrated in these vesicles in order to promote the formation of ACC nanoparticles, which are successively deposited into the calcification compartment where crystallization occurs 14 . The regulation of endocytosis and vesicular transport between membrane-bound cellular compartments is therefore strictly necessary in coral calcification, and the identification of genes related to these pathways, among the Tubastraea spp. aboral-specific genes, further underlines their importance also in non-symbiotic scleractinian species.
KEGG analysis further allowed us to identify a list of candidates that could play a role in calcification related ion transport ( Table 2). The list includes several genes belonging to the ammonium transporter family (AMT/ Rh/MEP), notably, AMT and Rh homologs. As well as for AMT transporters, also Rh transporters have been suggested to be involved in coral calcification. Rh homologs have been identified in the calicoblastic epithelium of the symbiotic scleractinian coral Acropora yongei, where they have been suggested to mediate a possible pathway for CO 2 -a critical substrate for CaCO 3 formation-in the ECM 101 . The identification of these genes also among the Tubastraea spp. aboral-specific ones strongly suggests a direct role of these transporters in non-symbiotic scleractinian calcification.
We also identified a large number of transporters belonging to the SoLute Carrier (SLC) families that, in vertebrates, constitute a major fraction of transport-related genes 102 (Table 2). Some of these members (SLC7, SLC25 and SLC35) have been previously reported to be involved in coral thermal stress, while others (SLC26) have been proposed to participate in coral larval development 103 , as well as cellular pH and bicarbonate metabolism 31 .
Two plasma-membrane homologs belonging to the SLC13 family have also been identified (Table 2). These transporters function as Na + -coupled transporters for a wide range of tricarboxylic acid (TCA) cycle intermediates 104 , and have been widely described in vertebrates for their role in calcification [105][106][107][108] . The tricarboxylic acid citrate has also been found to be strongly bound to the bone nanocrystals in fish, avian, and mammalian bone 109 , whereas in corals no study has shown the presence of citrate in the skeleton. In invertebrates, SLC13 members have been mainly described for their role in nutrient absorption 110,111 , as they provide TCA cycle metabolites, that are used for the biosynthesis of macromolecules, such as lipids and proteins 2,20 . These macromolecules are among the principal components of the skeletal organic matrix and SLC13 members might contribute to their transport into the coral aboral tissues.
SLC16 family members, and precisely the monocarboxylic acid transporters (MCTs), are also enriched in the total colony compared to the oral fraction ( Table 2). Members of the SLC16 family comprise several subfamilies that differ in their substrate selectivity 112 . In corals and sea anemones, SLC16 subfamilies transporting aromatic amino acids have been mostly characterized for their role in nutrient exchange between the coral host and its symbionts 36,113 , while no information is available for those transporting monocarboxylic acids. In human, MCTs function as pH i regulatory transporters by mediating the efflux of monocarboxylic acid (predominantly lactate) www.nature.com/scientificreports/ and H + , in tissues undergoing elevated anaerobic metabolic rates 114,115 , and in Tubastraea spp. they might be involved in H + extrusion at the sites of calcification perhaps functioning as pH i regulators. Interestingly, members of the SLC23 family (FC = 916.1), which comprise ascorbic acid transporters, are the most enriched in the total colony compared to the oral fraction, in Tubastraea spp. (Table 2). This result is in agreement with another RNA-seq study performed on swimming and settled larvae of the coral Porites astreoides, also showing that an SLC23 transporter is among the most highly expressed ion transporters in larvae initiating calcification 116 . Ascorbic acid is an essential enzyme cofactor that participates in a variety of biochemical processes, most notably collagen synthesis 117,118 . Collagen is a fibrillar protein, that forms one of the main components of extracellular matrices 119 . Previous studies have shown that the addition of ascorbic acid stimulates collagen production in many metazoans [120][121][122] , including corals 119 . It is thus possible that SLC23 transporters might provide ascorbic acid to the aboral tissues, and potentially the calcifying cells, which use it to promote collagen that, together with other ECM proteins, builds a structural framework for the recruitment of calcium binding proteins, as previously suggested 48,[123][124][125] .
Last but not least, our study also identified so-called "dark genes", i.e., genes that lack annotation 126 , within the list of aboral-specific genes. These genes are potentially equally important, as they are expressed in the aboral tissues along with other genes with known functions in calcification. It is therefore possible that "dark genes" and calcification-related genes may be linked, as they can be involved in the same pathway e. g. enzymes and/ or regulatory factors. Heterologous expression of "dark genes" in model systems, easy to manipulate and with available molecular tools for visualizing gene expression and protein localization (e. g. Nematostella vectensis) could be taken in consideration to investigate their role and contribute to functional annotation 127 .

Conclusion
The Tubastraea spp. transcriptome here provided is a fundamental tool which promises to provide insights not only about the genetic basis for the extreme invasiveness of this particular coral genus, but also to understand the differences between calcification strategies adopted by symbiotic and non-symbiotic scleractinian corals at the molecular level. The analysis of the aboral-specific genes of Tubastraea spp. revealed numerous candidates for a potential role in scleractinian calcification, including both previously described candidates (SLC4-γ, AMT-1like) and novel ion transporters (SLC13, −16, −23, and others) (Fig. 7). Future studies will then be required to better dissect the precise mechanisms behind these candidates and may offer further knowledge which could lead to the development of novel biotechnological strategies for prevention, management, and control of this and other invasive species.

Methods
Biological material and experimental design. Experiments were conducted on non-symbiotic corals belonging to the Tubastraea genus. Corals belonging to this genus possess poorly defined taxonomic features and several unidentified morphotypes that severely challenge species identification 128  Micro-dissection, RNA isolation and sequencing from Tubastraea spp. Three biological replicates of Tubastraea spp. were micro-dissected by separating the oral fraction from the total colony. Then, RNA was extracted from each fraction, as previously described 47 . Preparation of mRNAs, fragmentation, cDNA synthesis, library preparation, and sequencing using Illumina HiSeq™ 2000 were performed at the King Abdullah University of Science and Technology (KAUST) 93 .
Data analysis pipeline. Data analysis pipeline contained three major sections including: raw data preprocessing, de novo transcriptome assembly and post-processing of the transcriptome. First, raw reads of six individual libraries were subjected to quality trimming, using the software Trimmomatic (version 0.36) 129 . This step consisted in trimming low quality bases, removing N nucleotides, and discarding reads below 36 bases long. Then, contaminant sequences were removed, using the software BBDuk 130 , by blasting raw reads against a previously created contaminant_DB of the most common contaminant species-including Symbiodiniaceae. Clean and trimmed reads from all samples were then pooled together and further assembled using Trinity software (version 2.8.0) with default parameters 131 . The in-silico normalization was performed within Trinity prior to de novo assembly. To obtain sets of non-redundant transcripts, we applied the following filtering steps: (1) transcripts with more than 95% of identity were clustered together using CD-HIT software 132 and (2) all likely coding regions were filtered by selecting the single best open reading frame (ORF) per transcript, using TransDecoder (version 3.0.0) 133 . Also, in the latter step, transcripts with ORFs < 100 base pairs (bp) in length were removed before performing further analyses. The final transcriptome (referred to as transcriptome_all) was subjected to quality assessment via generation of ExN50 statistics, using "contig_ExN50_statistic.pl", and examination of orthologs completeness, using BUSCO (version 3) against eukaryota_odb10 database 134 . Transcriptome_all was then aligned against NCBI's non-redundant metazoan databases using Blastx 135 , with a cutoff E-value of < 10 -15 , and the alignment results were used to annotate all the unigenes (= uniquely assembled transcripts). For their further annotation and classification, OmicsBox software (version 2.0.36) 136 was used to assign Gene Ontology (GO) terms 137  www.nature.com/scientificreports/ Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis 139 . Additionally, differential abundance analysis to identify differentially expressed genes (DEGs) was performed using OmicsBox 136 . To convert the RNA-Seq data into quantitative measure of gene expression, we calculated the number of RNA-Seq reads mapping to transcriptome_all. Transcripts that had at least a log fold change (LogFC) of ± 1 with a false discovery rate (FDR or adjusted p-value) less than 0.05 were considered as differentially expressed.

Data availability
All data needed to evaluate the conclusions in the paper are present in the manuscript and/or the Additional Files. Additional data related to this manuscript may be requested from the authors. Genomic and transcriptomic data were obtained from the public available database of the National Center for Biotechnology Information or from the private database of the Centre Scientifique de Monaco.   29-34 (1999).